Two major isozymes of hexokinase have been found to occur in the hepatopancreas and muscles of Homarus americanus (lobster). They have been separated from each other by DEAE-cellulose ion exchange chromatography and by polyacrylamide gel electrophoresis and their kinetic properties and substrate specificity studied. One resembles type I and II hexokinases occurring in all vertebrates, having a very low Km for glucose and mannose and a high Km for fructose and being subject to product inhibition or control by low concentrations of ADP or glucose-6-phosphate. The other differs significantly, so far as we are aware, from any hexokinase which has previously been described. Phosphorylation of glucose is catalyzed only at very high glucose concentrations and is not inhibited by glucose-6-P. In this it resembles type IV hexokinase of vertebrates but it differs from that enzyme in its great affinity for fructose, mannose and 2-deoxy-glucose.